Specific binding assays have found widespread application in the fields of biomedical research and clinical diagnostics where they are used to determined the presence or encountered of a variety of substances (analytes) commonly encountered in biological fluids. Such substances may include proteins, drugs, hormones, vitamins, microorganisms, etc. In addition, specific binding assays may find utility in other fields, such as food processing and environmental quality control, for the detection of microorganisms and their toxins, or for detecting organic wastes.
Specific binding assays are commonly divided into homogeneous and heterogeneous assays. In a homogeneous assay, the signal emitted by the bound labeled component is different from the signal emitted by the unbound labeled component. Hence, the two can be distinguished without the need for a physical separation step. The classical homogeneous specific binding assay is the enzyme-multiplied immunoassay technique (EMIT), described in U.S. Pat. No. 3,817,837, issued to Rubenstein.
Homogeneous specific binding assays are rapid and easy to perform, either manually or with automated instruments. However, these tests typically require sequential additions and mixing, with intervening incubations, of sample plus antibody, then enzyme-analyte conjugate, followed by enzyme substrate color developer solution. Automation has been achieved with various types of analyzers including discrete (e.g. DuPont aca.TM.), centrifugal (e.g. Roche Cobas Bio.TM.), and linear flow (e.g. Technicon SMAC.TM.) However, homogeneous assays have several disadvantages: they are typically limited to detection of low molecular weight compounds, are prone to interferences, and are generally limited in sensitivity to detection of approximately 1 nanomolar analyte.
In heterogeneous assays, both large and small analytes can be detected, but the signal emitted by the bound and unbound labeled components is identical, hence the two must be physically separated in order to distinguish between them. The classical heterogeneous specific binding assay is the competitive radioimmunoassay (RIA), described by Yalow (Science 200:1245, 1978). Other heterogeneous binding assays are the radioreceptor assay, described by Cuatrecasas (Ann. Rev. Biochem. 43: 109-214, 1974), and the sandwich radioimmunoassay, described by Wide (pp. 199-206 of Radioimmunoassay Methods, Edited by Kirkham and Hunter, E. & S. Livingstone, Edinburgh, 1970). Because interferences are usually eliminated, and because excess binding reagents can sometimes be used, heterogeneous binding assays can be significantly more sensitive and reliable than homogeneous assays.
In a typical "double antibody" competitive RIA, a known amount of radiolabeled ligand and ligand present in the sample compete for a limited amount of antibody. Sufficient time is allowed for specific binding to occur, after which the antibody and bound ligand are precipitated by addition of anti-immunoglobulin, washed to remove unbound label by repeated centrifugation, and the amount of labeled ligand present in the bound phase is determined.
A sandwich assay can be used to achieve greater sensitivity for analytes such as antigen in an immunoassay. In such an assay, excess ligands are used to force binding at concentrations below the dissociation constant of the binding pair. In the typical sandwich immunoassay, two antibody types are required, each of which can bind simultaneously to the antigen. One antibody is bound to a solid phase, while the other is labelled. As with competitive RIAs, one or more discrete washing steps to separate bound and unbound label are required, and sequential addition of reagents is typical.
Because the solid phase must be isolated and washed, and because sequential reagent additions are frequently required, heterogeneous assays tend to be time consuming and labor-intensive. However, they work equally as well for low and high molecular weight compounds, are less prone to interferences than homogeneous assays, and can be sensitive to sub-picomolar antigen concentrations. Automation of heterogeneous immunoassays has been accomplished (ARIA II.TM. by Becton Dickinson, CentRIA.TM. by Union Carbide), but this has required either sophisticated and expensive instrumentation to carefully control liquid flow and to monitor bound and unbound fractions, or it has resulted in the detection only of the unbound label flowing through a rapidly hydrated antibody solid phase.
Several attempts have been made to eliminate the inconvenience of washing steps in heterogeneous binding assays. For example, Glover et al., GB 1,411,382, describe a method for measuring the amount of unbound radiolabel, after partial separation from bound label, by shielding the bound (lower) phase. However, it is well known in the art that the sensitivity and precision of specific binding assays is severely limited if changes in the unbound rather than the bound labeled component are measured. Furthermore, methods which lack a washing step have the disadvantage of detecting both tightly binding (specific) and weakly binding (nonspecific) label, resulting in very high nonspecific signal. Charlton et al., U.S. Pat. No. 4,106,907, issued Aug. 15, 1978, disclose another container for radioactive counting which consists of a tapered reaction tube having a radiation shield extending up from the bottom of the tube to a uniform height, such that only radiation from the supernatant (the unbound labeled fraction) can be detected. This method is subject to the same limitations as Glover et al., supra.
Chantot et al., Analyt. Biochem. 84:256, 1978, describe a radioreceptor assay method for measuring the binding of radiolabeled ligands to membrane receptors. The technique involves counting the total amount of radiolabel present, centrifuging the sample, and recounting with an externally mounted copper screen which serves to absorb radiation from a defined volume of the supernatant. The screen itself consists of a copper sleeve mounted on the outside of a custom-made test tube having a small knob precisely positioned above the base to support the screen. This method suffers from the disadvantage of requiring double detection, and suffers as well from high nonspecific binding as described above for the Glover and Charlton methods. Furthermore, the tube is vulnerable to jamming and breakage in standard gamma counters. As with the above-described "screening" methods, the large diameter of the screen allows significant scattered radiation from within the screened volume to impinge on the detector, resulting in inaccurate measurements of the unscreened label. Also, because bound label is directly adjacent to and in contact with unbound label, normal and unavoidable variability in the position of the screen or in the volumes of the unbound and bound phases can cause significant variability in signal.
Bennett et al., (J. Biol. Chem. 252: 2753, 1977) describe a radioreceptor assay in which, after mixing and incubating reagents, the assay mixture is transferred to a centrifuge tube to wash the solid phase containing bound label. They employed prolonged (30minutes) high speed centrifugation to force the solid phase into a solution of 20% sucrose, followed immediately by freezing the assay tube in liquid nitrogen and excising the tip of the tube containing the solid phase and bound label. This method provides more effective separation of bound and unbound label than those described above, but has several significant disadvantages. The assay mixture cannot be incubated in situ on top of the sucrose solution, thus requiring separate incubation and separation vessels, because reactants would diffuse into the solution. Care must be used in loading the assay mixtures onto these sucrose solutions because mixing will cause dilution of the assay mixture, thus changing the equilibrium for assay reactants. The separation is relatively lengthy, and assay tubes must be frozen immediately after centrifugation because the bound label can dissociate from the solid phase and diffuse away from the tip of the separation tube. Finally, excising the tip of separation tubes is inconvenient, time-consuming, difficult to perform reproducibly, exposes the user to the risk of liquid nitrogen burns and radioactive contamination from fragments of frozen tubes and their contents, and would be very difficult to automate.
In U.S Pat. No. 4,125,375 (issued Nov. 14, 1978), Hunter describes a method and automated instrumentation for performing heterogeneous immunoassays by carefully injecting a sucrose solution underneath a previously equilibrated immunoassay mixture containing particles of higher density than the sucrose solution. The particles are allowed to settle through the injected subphase, thereby separating the particles from the unbound label. This method potentially eliminates some of the disadvantages inherent in the Bennett et al. method, but suffers from several significant shortcomings. These shortcomings include that: (1) it requires separate preequilibration of the assay mixture prior to separation of bound and free label, plus removal of liquid waste, and thus cannot be self-contained, (2) the method is not readily adaptable to the most rapid (centrifugal) separations, (3) it suffers from potential dilution and diffusion artifacts as in the Bennett et al. method, (4) it is not suitable for convenient and reproducible manual assays, and (5) any automated instrument would require plumbing for reagent delivery and waste disposal.
Linsley et al., Proc. Natl. Acad. Sci. (USA) 82: 356, 1985, describe a radioimmunoassay for type I transforming growth factor using S. aureus in which the bound label is separated from the unbound label by rapid sedimentation into a solution of 10% sucrose, followed by freezing in liquid nitrogen and excision of the tip of the centrifuge tube to determine the sedimented bound label. This method is essentially an immunoassay embodiment of the radio-receptor assay described by Bennett et al., with the inherent disadvantages of the former method.
Although each of the methods described above have brought minor improvements to the state of the art, there remains a need in the art for a method of specific binding assay which combines the ease and rapidity of homogeneous techniques with the enhanced sensitivity typical of heterogeneous techniques, for both isotopic and nonisotopic applications, without the undesirable variability, delay, labor, and dissociation which occur during the wash steps. Further, the method should allow rapid separations, should be convenient for manual use with standard detection instruments, and should be readily adaptable to semiautomated or fully automated instrumentation. Ideally the method should be self-contained, have minimal plumbing and moving parts, and be compatible with fully predispensed reagents. The present invention fulfills this need, and further provides other related advantages.